Peripherin is tyrosine-phosphorylated at its carboxyl-terminal tyrosine.
Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim- cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim- cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim- cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.
Angelastro
J M
JM
Department of Pathology and Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.
Ho
C L
CL
Frappier
T
T
Liem
R K
RK
Greene
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007381:Intermediate Filament Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008562:Membrane Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016716:PC12 Cells; D064531:Peripherins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012584:Sciatic Nerve / Q000378:metabolism*; D018411:Spodoptera; D014162:Transfection
D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C578994:PRPH protein, human; D064531:Peripherins; D011994:Recombinant Proteins; D019000:Phosphotyrosine